The present invention relates to a Mirabilis Antiviral Protein (to be referred to as MAP hereinafter) variant and, more particularly, to a MAP variant whose inhibition activity in in vitro protein synthesis is improved as compared with that of natural MAP.
2. Description of the Related Art
The present inventors previously separated a novel basic protein from Mirabilis jalapa and found that this protein exhibited antiviral activity against a plurality of plant viruses. This protein was called MAP and was claimed in Published Examined Japanese Patent Application No. 63-61317. All amino acid sequences, synthesis of a gene based on the specified amino acid sequences, and construction of a system in E. coli, in which MAP is produced and secreted into the medium using this complete or full synthetic MAP gene were claimed in Published Unexamined Japanese Patent Application No. 2-186988 and Japanese Patent Application No. 1-210767.
The MAP is a type of ribosome inactivating protein (RIP) widely existing in plants and microorganisms, and exhibits an RNA N-glycosidase activity with high specificity for ribosomal RNA as a substrate. Ribosomes are inactivated by this activity, inhibiting protein synthesis, as is well known. Such protein synthesis inhibition activity is highly toxic in cells. In recent years, this toxicity has been utilized to develop immunotoxins having high selectivity, for example, by linking a ricin A chain, a type of RIP derived from Ricinus communis, to various antibodies. These immunotoxins are utilized, for example, in missile therapy.
This protein toxin possesses high antigenicity. There exists the possibility that an antibody against the toxin is produced in living bodies. Thus, long-term doses may adversely affect the living body. Therefore, proteins possessing various properties for use as toxic proteins are required.
Under these circumstances, the MAP is one of the most promising candidates as a toxin for an immunotoxin. The protein synthesis inhibition activity in a rabbit reticulocyte system has been shown to be only about 1/30 of the ricin A chain.
Protein engineering involving gene manipulation techniques has developed remarkably in recent years. In various applications, amino acid sequences of natural proteins are altered to produce proteins whose inherent activities are modified. It has been found in these applications that disulfide bonds (S-S bonds) in protein molecules are closely associated with the flexibility of the protein molecule, and that the activity can be greatly changed, depending on the presence or absence of S-S bonds (Matsumura et. al., Nature 342, pp. 291-293 (1989); Matsumura et. al., Proc. Natl. Acad. Sci. USA 87, pp. 6562-6566 (1989); Kozo Hamaguchi, Biochemistry 1, pp. 1-13 (1991); and Pace, Biotrend 2-4, pp. 105-110 (1990)).
When the foregoing is taken into consideration, the production of proteins possessing greater activity and, more particularly, possessing greater protein synthesis inhibition activity is expected by utilizing MAP genes.